Salivary gland (SG) hypofunction is a predictable and highly debilitating consequence of the use of ionizing radiation in the treatment of head and neck cancers, which results from irreversible damage to salivary acinar cells. Clinically, the currently accepted treatment for radioprotection of salivary glands involves the use of amifostine to protect normal salivary tissue against radiation and cytotoxic damage. This is associated with significant side effects and is of uncertain effectiveness in combination radio/chemotherapy regimens. Amifostine is thought to exert its radioprotective effects through the activation of the survival- promoting transcription factor, nuclear factor ?B (NF?B), which regulates anti-oxidant genes, and also opposes the action of pro-apoptotic mediators. The central hypothesis of this proposal is that targeted delivery to salivary gland cells of selected siRNAs that either engage cell survival pathways known to play a role in radioprotection, or block the action of mediators of irradiation-induced cell death, will result in the protection of these cells from irradiation-induced damage. We propose two Specific Aims: 1) to develop efficient, nanoparticle-based methods to deliver siRNA to SG cells. We will use well-characterized peptide ligands to target siRNA-containing nanoparticles to SG cells, including (i) cell-permeabilizing peptides (CPP) and (ii) peptide ligands for apical integrins. These in vitro experiments will use cultured SG cells and a panel of different siRNA-containing nanoparticle formulations. Successful siRNA delivery will be assessed using fluorescently labeled siRNA molecules and also by measuring knockdown of a reporter gene (GFP);and 2) to determine whether siRNA-mediated gene knockdown can protect against irradiation-induced SG damage. Two classes of siRNA molecules will be tested for their ability to protect salivary gland tissue against irradiation-induced damage: (i) siRNAs that will transiently activate the survival promoting factor, NF?B (mimicking the action of amifostine);and (ii) siRNAs that will transiently inhibit the expression of pro-apoptotic mediators that are induced following radiation treatment (PKC4, Bax and p53). Following in vitro characterization of siRNA function, experiments will be conducted in vivo to determine the effect of siRNA-mediated gene knockdown on irradiation-induced cell damage using two radiation models: (a) acute induction of SG cell apoptosis following exposure of mice to a low dose of irradiation, and (b) chronic induction of xerostomia and acinar depletion following exposure of mice to a large radiation dose. It is expected that these experiments will provide proof-of-principle support for our hypothesis that siRNA-mediated gene knockdown can protect against irradiation-induced damage to salivary gland tissue. Project Narrative: Salivary gland tissue is highly sensitive to ionizing radiation, and can become permanently damaged as a result of cancer radiotherapy. The resultant loss of saliva production leads to a permanent and profound reduction in quality of life, with severe oral complications. The present proposal seeks to develop a new and more effective approach to the treatment of this predictable and important consequence of cancer therapy.